Mechanism of peiminine in inhibiting malignant biological behaviors of glioma cells based on transcriptomics

MAO Xingyun; GUO Lingdi; WANG Lifeng; LI Ruiguo; WANG Yiming; HENG Xueyuan; LI Li

doi:10.13431/j.cnki.immunol.j.20250120

Abstract：Objective To evaluate the inhibitory effect of the natural product peiminine on malignant biological behaviors in human glioma cells and to elucidate its mechanisms based on transcriptomic analysis.Methods The effect of different concentrations of peiminine on the viability of human glioma cell lines U251 and LN229 was assessed by CCK-8 assay to determine half-maximal inhibitory concentration(IC50)values.IC50-adjacent concentrations(80 μmol/L for U251 and 110 μmol/L for LN229)were used as effective intervention concentrations for subsequent experiments.Effects of peiminine on cell proliferation,migration,and invasion were evaluated by colony formation,cell scratch wound healing,and Transwell assays,and apoptosis was analyzed by flow cytometry.Transcriptome sequencing of U251 cells treated with peiminine was conducted to identify differentially expressed genes(DEGs)for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses.The key gene RHOU expression was validated by Western blot analysis.A stable RHOU knockdown cell line was established and treated in combination with peiminine.The role of RHOU in peiminine-induced apoptosis was investigated using flow cytometry.Gene set enrichment analysis(GSEA)and quantitative real-time PCR(RT-qPCR)were performed to verify the regulatory effects of RHOU on key downstream components of the E2F signaling pathway.Results Peiminine significantly inhibited the viability of U251 and LN229 cells in a dose-dependent manner,effectively suppressed the colony formation,migration,and invasion capabilities of glioma cells,and markedly induced cellular apoptosis.Transcriptomic analysis identified 1,507 DEGs primarily enriched in pathways related to cell cycle,cell proliferation,apoptosis,and cell adhesion.Among these,RHOU was significantly downregulated among the DEGs.Western blot confirmed significantly decreased RHOU protein expression after peiminine treatment.RHOU silencing enhanced peiminine-induced pro-apoptotic effect.GSEA based on RHOU expression showed significant enrichment of the E2F signaling pathway.RHOU knockdown significantly downregulated the mRNA expression levels of key E2F pathway genes,including E2F1,CCND1,and CCNE1.Conclusion Peiminine significantly downregulated the expression of key gene RHOU,thereby inhibiting the activity of the downstream E2F signaling pathway and ultimately blocking the malignant biological progression of glioma cells.